This is a resubmission of a renewal application for a second five years of funding for ?Role of Retromer-mediated Retrograde Transport in HPV Entry,? 5R01-AI102876. Despite effective vaccines, HPV infection and HPV-induced diseases remain a major public health problem worldwide. HPV is the most common sexually transmitted virus and is responsible for 5% of all cancer, millions of cases of genital warts, and countless cases of other types of cutaneous and mucosal warts (most caused by non- vaccine HPV types). There are no specific treatments for HPV. With the support of this grant, we have made important contributions to understanding intracellular trafficking of HPV during entry. We discovered the central role of retrograde transport in HPV infection, showed that retromer is required for sorting of the incoming virus particle into the retrograde pathway and that HPV is a new class of retromer cargo, and discovered a novel cell-penetrating peptide (CPP) on the L2 capsid protein that drives it into the cytoplasm to engage the retromer. This is the first example where retromer or a CPP has been shown to play a role in virus entry. These results have been published in top journals and have fundamentally changed our understanding of HPV entry and challenged dogma concerning the biological roles of retromer and CPPs and the definition of transmembrane proteins. Here, we will study how the L2 protein accomplishes these amazing feats. We will determine the sequence and physiological requirements for L2 protrusion using a novel split-GFP assay we developed. We will test our hypothesis that the abundance of CPP sequences in the extant papillomavirus virome reflects their membrane- penetrating activity and we will establish how sequences flanking the core CPP modulate its activity. We will establish the nature and extent of L2 membrane protrusion and determine if L2 truly adopts a TM orientation, map L2 segments exposed in the cytoplasm, and test whether it protrudes through the membrane sequentially. We will continue to isolate and characterize artificial small TM proteins that inhibit HPV entry and use them to identify novel HPV entry factors and dissect their contribution to retrograde trafficking, and we will exploit our new-found mechanistic understanding of HPV entry to design inhibitory peptides that harness the membrane-penetrating activity of CPPs to deliver the retromer binding site into the cytoplasm where it displaces retromer from incoming HPV. These experiments will elucidate important mechanistic aspects of HPV entry, lead to the development of new approaches to prevent and treat HPV infection, and provide important new insights into fundamental cell biology.